Blood Res.  2020 Jun;55(2):91-98. 10.5045/br.2020.2020031.

Natural soluble human leukocyte antigen class I in donor serum neutralizes donor-specific HLA alloantibodies in recipient serum

• Won DI
• Lee NY
• Lim JH
• Han YS
• Kim CD
• Huh S

• KMID: 2503483
• DOI: http://doi.org/10.5045/br.2020.2020031

Abstract

Background
Human leukocyte antigen (HLA) molecules are cell-bound but can be identified in a soluble form. These soluble HLA (sHLA) molecules have an immunomodulatory function. We investigated whether natural sHLA in donor serum can neutralize donor-specific HLA alloantibodies (DSAs) in recipient serum.
Methods
Neutralizing effects of donor serum on DSAs in recipient serum were measured using inhibition assay principle of flow cytometric crossmatch (FCXM), performed using sera from 143 kidney transplant recipients and their donors. The adding of donor serum to recipient serum yielded lower mean fluorescence intensity (MFI) ratios (test/control) than when diluent was added [Roswell Park Memorial Institute (RPMI) or third-party serum], which was presumed to be caused by the neutralizing effects of sHLA.
Results
In the recipient group with class I DSAs alone (N=14), donor serum addition to recipient serum resulted in lower T cell MFI ratios [2.25 (1.31‒32.51)] than those observed on RPMI addition [3.04 (1.33‒125.39), P ＜0.05]. In the recipient group with class II DSAs alone (N=27), donor serum addition showed no significant difference in B cell MFI ratios [5.03 (1.41‒103.53)] compared to diluent addition: RPMI [4.50 (1.34‒145.98)] or third-party serum [5.08 (1.44‒138.47)], P ＞0.05 for both.
Conclusion
Using inhibition FCXM, we verified that natural sHLA class I in donor serum neutralizes DSAs in recipient serum. However, no neutralizing effects of sHLA class II were revealed in this study. These potentially beneficial effects of sHLA infused via blood-derived products should be considered when desensitizing highly HLA-sensitized patients.

Keyword

Soluble HLA; Donor-specific HLA alloantibody; Neutralization; Flow cytometric crossmatch

Figure

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  Fig. 1 Data analysis of FCXM. T cells were gated using lymphgate R1 and T cell gate R2. B cells were gated using lymphgate R1 and B cell gate R3. On the anti-IgG FITC histogram of the gated T cells or B cells, the geometric mean of the peak within the marker M1 was obtained and used to calculate the test/control MFI ratio. Abbreviations: AB, group AB serum from healthy individuals; DC, donor cells; FITC, fluorescein isothiocyanate; FSC, forward scatter; MFI, mean fluorescence intensity; PE, phycoerythrin; PerCP, peridinin chlorophyll protein complex; RS, recipient serum; SSC, side scatter.

• 

  Fig. 2 Preparations of “diluted” or “mixed” serum and paired comparisons among them. Recipient serum was mixed with an equal volume of donor serum (mixed serum) and incubated at 25°C for 30 min. For comparison with mixed serum, the recipient serum was diluted with an equal volume of diluent, either RPMI or third-party serum (diluted serum). The green symbols resembling an uppercase letter Y represent antibodies, and the small round or triangular symbols represent sHLA. In the mixed serum, the sHLA (green round dots) from a donor (previous sensitizer) can neutralize donor-specific antibodies (green “Y”) in recipient serum.

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  Fig. 3 Representative diagram of neutralizing effects superior to matrix effects on the anti-IgG histogram of gated T cells in FCXM. T cell peaks are shown for mixed serum (donor+recipient), diluted serum (with either RPMI or third-party group AB serum), and neat serum. The peak for the neat serum in conventional FCXM appears in the right-most position, reflecting its original DSA level. In the diluted serum, the dilution and matrix effects are owing to the decreased DSA and immunoglobulin concentrations, respectively. Neutralizing effects in the mixed serum are evident only when they surpass the matrix effects in the corresponding diluted serum (MFI, diluted>mixed). In contrast, when there are no neutralizing effects, only matrix effects are apparent (MFI, mixed>diluted). Abbreviations: DSA, donor-specific HLA alloantibody; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; sHLA, soluble human leukocyte antigen.

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  Fig. 4 Dilution effects on inhibition FCXM with RPMI as the diluent. In principle, the MFI ratio of a serum diluted with RPMI should be less than that of the neat serum with DSAs, and thus be plotted under the diagonal line, y=x. Most MFI ratios of diluted sera in the inhibition FCXM were lower than those of neat sera in the conventional FCXM. The higher values might be due to the prozone phenomenon (excess antibody). Abbreviations: DSA, donor-specific HLA alloantibody; FCXM, flow cytometric crossmatch; MFI, mean fluorescence intensity.

• 

  Fig. 5 The neutralizing effects of donor sera compared to third-party sera as diluents in inhibition FCXM. When the neutralizing effects of the sera of previous sensitizers are evident, their MFI ratios should be less than those generated by the sera diluted with third-party sera and thus should be plotted under the diagonal line, y=x. In the group with both class I and class II DSAs (N=33), neutralizing effects were evident in both T cell and B cell FCXM. In the group with any positive DSAs (class I or class II DSAs, N=74), neutralizing effects were evident in the B cell FCXM only. Abbreviations: DSA, donor-specific HLA alloantibody; FCXM, flow cytometric crossmatch; MFI, mean fluorescence intensity.

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